GSM6813200: 5310_2_Pool_of_GAS_cells_in_vivo_and_in_vitro_2_CTCCTAGT_S10; Homo sapiens; RNA-Seq
Harvested tumors from NSG-B2m-/- mice were manually minced and enzymatically digested in RPMI medium (Gibco) supplemented with 5 Wünsch units/ml TH Liberase/ml (Roche) 25 μg/ml DNase I (Roche) for 20 min at 37 °C under continuous shaking. Subsequently, cell digests were filtered through a 70-μm strainer (Falcon) and single-cell suspensions were stained with IR-Dye (Invitrogen) and TotalSeq Hastag antibodies (TotalSeq-A or B, Biolegend). Cells from each sample were combined at equal numbers, and CFP+ (i.e. bystander) tumor cells were sorted from this cell pool on a FACSaria Fusion (BD biosciences). Sorted single cell suspensions were diluted to a final concentration of 1,000 cells/μl in 1xPBS containing 0.04% weight/volume BSA. The Chromium Controller platform of 10X Genomics was used for single cell partitioning and barcoding. Per single cell suspension, each cell's transcriptome was barcoded during reverse transcription, pooled cDNA was amplified and Single Cell 3' Gene Expression libraries and Cell Hashing libraries via Feature barcode technology were prepared, according to the manufacturer's protocol (CG000183, CG000206 and CG000317, 10X Genomics). All libraries were quantified on a 2100 Bioanalyzer Instrument following the Agilent Technologies Protocol (Agilent DNA 7500 kit, G2938-90024). Sequence library pools were composed and quantified by qPCR, according to the KAPA Library Quantification Kit Illumina® Platforms protocol (KR0405, KAPA Biosystems) HiSeq 2500 Illumina sequencing was used for paired-end sequencing of the Single Cell 3' Gene Expression library and Cell Hashing libraries, respectively, at a sequencing depth of between 20,000-60,000 reads /cell and approximately 3,500 genes/cell. HiSeq 2500 paired-end sequencing was performed using 100 cycles for Read 1, 8 cycles for Read i7, and 100 cycles for Read 2, using HiSeq PE Cluster Kit V4 (PE-401-4001, Illumina) and multiple HiSeq SBS Kit V4 50 cycle kits (FC-401-4002, Illumina)